PROJECT SUMMARY Although many technologies exist for studying protein structures, none possess a combination of speed, selectivity, accuracy, resolution, and flexibility. The long-term goal is to use a suite of gas-phase chemistries for ion/ion reactions coupled to ion mobility (IM) and tandem mass spectrometry (MS) measurements for high- throughput, virtually sample-prep free, protein structure measurements. The overall objective for this exploratory project, which is the next step toward attaining our long-term goal, is to implement rapid ion/ion cross-linking reactions on an IM/MS platform, using tandem mass spectrometry to identify cross-linked sites. The rationale for the development of this technology is to combine the information from native IM/MS with information obtained from cross-linking in an experimental method conducted on the sub-second timescale. The increase in throughput, information, and lack of sample prep (compared to, e.g., X-ray diffractometry, nuclear magnetic resonance, or cryo-electron microscopy) is expected to advance biomedical research determining protein structure and function to where protein structural determinations can become rapid and routine. The overall objective of this application will be reached through the following Specific Aims: 1. Combine gas-phase ion/ion cross-linking of intact proteins with IM/MS measurements; and 2. Use IM combined with tandem MS to determine cross-linking locations for intact proteins. For the first aim, a variety of monomeric and multimeric proteins will be cross-linked in the gas-phase. Changes in overall structure between cross-linked and unmodified proteins, as well as between solution and gas-phase cross-linked proteins, will be measured by IM. Under the second aim, a combination of collision induced dissociation (CID) and electron capture dissociation (ECD) will be used to determine cross-linked sites. The proposed technology is innovative because it represents a substantive departure from the status quo by coupling cross-linking and native IM/MS analysis into one gas-phase mass spectrometry experiment, allowing rapid cross-linking analysis and providing multiple complementary measures of gas-phase protein structure. This new technology is significant because it is expected to become a rapid tool for high-throughput characterization of primary, secondary, tertiary, and quaternary protein structure. When fully developed, the technology has the potential to be used for complex mixtures of intact proteins and for rapid screening of interactions with small molecules/drugs, creating new opportunities in clinical research, treatment, and drug design.